Li Na, Zhang Yiyi, Yao Lunguang, Shi Yunpeng, Zhao Qin, Huang Baicheng, Sun Yani
Key Laboratory of Ecological Security, Collaborative Innovation Centre of Water Security for Water Source Region of Mid-line of South-to-North Diversion Project of Henan Province, Henan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, School of Life Sciences and Agricultural Engineering, Nanyang Normal University, Nanyang, China.
Shijiazhuang Customs (Huanghua Port), Cangzhou, China.
Front Microbiol. 2022 Jan 21;13:839845. doi: 10.3389/fmicb.2022.839845. eCollection 2022.
Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral ) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3'UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.
由于便于观察，携带报告蛋白作为工具的重组病毒被广泛应用于病毒生物学研究。此前，我们构建了一种带有增强型绿色荧光蛋白（EGFP）报告基因的重组致病性猪繁殖与呼吸综合征病毒（PRRSV），用于监测病毒传播和中和抗体筛选。带有不同类型报告基因的PRRSV可支持更多应用场景。在此，我们描述了一种新的高致病性PRRSV（HP-PRRSV）的遗传稳定感染性克隆，其携带DsRed（一种从珊瑚中分离的红色荧光蛋白）基因。在重组感染性克隆中，PRRSV的转录调控序列2（TRS2）插入开放阅读框7（ORF7）和3'UTR之间，以驱动DsRed基因的转录，这使其在病毒基因组中成为一个独立的转录单元。利用细菌人工染色体（BAC）系统和巨细胞病毒（CMV）启动子，成功拯救了插入DsRed的重组HP-PRRSV，并且在MARC-145细胞中与野生型病毒相比显示出相似的生长和复制模式。此外，DsRed蛋白在重组病毒中稳定表达至少10代，荧光强度和密度一致。利用带有DsRed蛋白的重组HP-PRRSV，通过活细胞成像观察了MARC-145中的病毒追踪情况。同时，通过流式细胞术对DsRed荧光阳性细胞进行定量，为检测PRRSV感染水平的标准方法提供了一种替代方法。这种表达DsRed荧光蛋白的重组PRRSV可能是病毒生物学基础研究的有用工具，并展示了在PRRSV中稳定表达外源基因的新设计。
